GLABRA1 (GL1) belongs to the group of R2R3-MYB transcription factors and is known to be essential for trichome initiation in Arabidopsis. In our previous study, we identified a GL1 ortholog in Brassica rapa as a candidate for the gene controlling leaf hairiness by QTL analysis and suggested that a 5-bp deletion (B-allele) and a 2-bp deletion (D-allele) in the exon 3 of BrGL1 and a non-synonymous SNP (C-allele) in the second nucleotide of exon 3 possibly cause leaf hairlessness. In this study, we transformed a B. rapa line having the B-allele with the A-allele (wild type) or the C-allele of BrGL1 under the control of the CaMV 35S promoter. The transgenic plants with the A-allele showed dense coverage of seedling tissues including stems, young leaves and hypocotyls with trichomes, whereas the phenotypes of those with the C-allele were unchanged. In order to obtain more information about allelic variation of GL1 in different plant lineages and its correlation with leaf hairiness, two GL1 homologs, i. e., RsGL1a and RsGL1b, in Raphanus sativus were analyzed. Allelic variation of RsGL1a between a hairless line and a hairy line was completely associated with hairiness in their BC1F1 population. Comparison of the full-length of RsGL1a in the hairless and hairy lines showed great variation of nucleotides in the 3′ end, which might be essential for its function and expression.